The cystic fibrosis transmembrane conductance regulator protein (CFTR) is a cAMP-activated chloride (Cl−) channel expressed in epithelial cells in mammalian airways, intestine, pancreas and testis. CFTR is the chloride-channel responsible for cAMP-mediated Cl31  secretion. Hormones, such as a β-adrenergic agonist, or toxins, such as cholera toxin, lead to an increase in cAMP, activation of cAMP-dependent protein kinase, and phosphorylation of the CFTR Cl− channel, which causes the channel to open. An increase in the concentration of Ca2+ in a cell can also activate different apical membrane channels. Phosphorylation by protein kinase C can either open or shut Cl−channels in the apical membrane. CFTR is predominantly located in epithelia where it provides a pathway for the movement of Cl− ions across the apical membrane and a key point at which to regulate the rate of transepithelial salt and water transport. CFTR chloride channel function is associated with a wide spectrum of disease, including cystic fibrosis (CF) and with some forms of male infertility, polycystic kidney disease and secretory diarrhea.
The hereditary lethal disease CF is caused by mutations in the gene encoding the CFTR protein, a cAMP-activated Cl− channel expressed in airway, intestinal, pancreatic, and other secretory and absorptive epithelia. The principal clinical problem in CF is recurrent lung infections resulting in progressive deterioration in lung function. The most common CFTR mutation, deletion of phenylalanine-508 (ΔF508-CFTR), is present in at least one allele in about 90% of CF patients (Egan et al., (2004) Science 304:600-602). ΔF508-CFTR causes CL− impermeability because it is not processed correctly, causing it to be retained at the endoplasmic reticulum (rather than the plasma membrane). ΔF508-CFTR also has reduced intrinsic Cl− conductance relative to wild type CFTR.
Strategies have been investigated to correct the defects in ΔF508-CFTR cellular processing and intrinsic function in cells. Cell growth at low temperature (<30° C.) (Denning et al., (1992) Nature 358, 761-764) or with high concentrations of chemical chaperones such as glycerol (Sato et al., (1996) J. Biol. Chem. 271, 635-638; Brown, et al., (1996) Cell Stress & Chaperones 1, 117-125) corrects partially defective ΔF508-CFTR cellular processing by a mechanism that may involve improved protein folding and stability (Sharma et al., (2001) J. Biol. Chem. 276, 8942-8950). A sustained increase in intracellular calcium concentration by thapsigargin also corrects defective ΔF508-CFTR processing (Egan et al., (2002) Nature Med. 8, 485-492), possibly by interfering with interactions with molecular chaperones.
Compounds like phenylbutryate facilitate ΔF508-CFTR cellular processing by altering chaperone function and/or transcriptional enhancement (Rubenstein et al., (2000) Am. J. Physiol. 278, C259-C267; Kang et al., (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 838-843). Although these approaches provide insight into mechanisms of ΔF508-CFTR retention at the endoplasmic reticulum, they probably do not offer clinically-useful therapies.
ΔF508-CFTR has significantly impaired channel activity even when present at the cell plasma membrane (Dalemans et al., (1991) Nature 354, 526-528). Cell-attached patch-clamp measurements showed reduced ΔF508-CFTR open channel probability and prolonged closed times even with maximal cAMP stimulation (Haws et al., (1996) Am. J. Physiol. 270, C1544-C1555; Hwang et al., (1997) Am. J. Physiol. 273, C988-C998). Patch-clamp measurements in excised membranes indicated 7-fold reduced ΔF508-CFTR activation after phosphorylation compared to wildtype CFTR. Relatively high concentrations of the flavone genistein (>50 μM, Hwang, et al., (1997) Am. J. Physiol. 273, C988-C998; Wang et al., (2000) J. Physiol. 524, 637-638) or the xanthine isobutylmethylxanthine (>1 mM, Drurnm et al., (1991) Science 254, 1797-1799) in combination with cAMP agonists increase ΔF508-CFTR channel activity. Again, these studies have not offered any clinically useful therapies.
There is accordingly still a need for compounds that can activate mutant CFTR, e.g., ΔF508-CTFR, and methods of using such compounds for the study and treatment of CF and the treatment and control of other secretory disorders. The present invention addresses these needs, as well as others.